Archive of SID

Original Article Middle East Journal of Cancer; October 2020; 11(4): 476-482

SS18-SSX Rearrangement in Synovial Sarcoma Using Fluorescent in Situ Hybridization: A Report from an Iranian Population and Literature Review

Akbar Safaei, Elham Jansar, Ahmad Monabati, Maral Mokhtari, Mehdi Montazer♦

Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract Please cite this article as: Safaei A, Jansar E, Monabat: Background: Synovial sarcoma is an aggressive soft tissue sarcoma. It has a wide A, Mokhtari M, Montazer M. spectrum of histopathologic patterns and uncertain immunohistochemistry, rendering SS18-SSX rearrangement in synorial Sarcoms using it a diagnostic challenge. The objective of this study was to investigate the diagnostic fluorescent in sita utility and impact of SS18-SSX rearrangement evaluation in an Iranian population hybridization: A report from previously diagnosed with synovial sarcoma without such molecular tests. an Iranian population and litarature review. Middle East J Method: We conducted this cross-sectional study on 44 formalin-fixed paraffin- Cancer. 2020;11(4): 476-82. embedded tissue blocks obtained from 23 synovial sarcoma patients (males, 69%; doi: 10.30476/mejc. mean age, 36.4 years) and 11 cases with other neoplasms as negative controls. We assessed these specimens for SSX-SS18 rearrangement by break-apart fluorescent in situ hybridization (FISH) probes. Results: FISH study showed SS18-SSX fusion gene in 17 (73.91%) cases while six (26.09%) cases and negative controls did not show SS18-SSX fusion. Histopathologic type of tumor was significantly related to the presence of rearrangement (P=0.002) (rearranged: 11 biphasic and six monophasic tumors; non-rearranged: three monophasic and three poorly-differentiated tumors). Conclusion: This study supports the idea that molecular studies contribute to the confirmation of synovial sarcoma diagnosis, particularly in monophasic and poorly- differentiated subtypes, which show vague immunohistochemical results.

Keywords: Synovial sarcoma, Gene fusion, Fluorescent in situ hybridization, SS18- SSX1 fusion

♦Corresponding Author: Mehdi Montazer, MD Introduction adolescents and young adults aged Department of Pathology, Synovial sarcoma is an aggressive 15-40 years. This condition impacts Faculty of Medicine, Setad Sq., Zand St, Shiraz, Iran malignant soft tissue tumor of males slightly more than females Tel/Fax: +98-71-32301784 uncertain type accounting for 5-10% (ratio, 1.2:1) and mainly occurs in Email: [email protected] of all soft tissue sarcomas.1,2 Synovial the extremities. However, it has been sarcoma is most prevalent in reported in all ages and a wide variety

Received: June 30, 2019; Accepted: January 05, 2020

www.SID.ir Archive of SID SS18-SSX Rearrangement in Synovial Sarcoma of locations. Moreover, synovial sarcoma shows who had undergone resection operation in the a broad spectrum of histological patterns ranging main hospitals of the Shiraz University of Medical from the more easily recognizable biphasic type Sciences (Nemazee, Faghihi, Chamran, Khalili with distinct epithelial and spindle cell parts to the and Madar-va-Kodak) between 2007 and 2016. challenging monophasic and poorly-differentiated Besides histomorphology, 16 cases also had types.1, 2 These facts have made synovial sarcoma immunohistochemistry. We reviewed all archived a usual suspect existing in the differential diagnosis slides, confirmed the diagnosis based on World list for almost all soft tissue sarcomas. Health Organization (WHO) classification, graded Immunohistochemistry, especially the finding the tumors according to French National of epithelial markers, CD99, and Bcl2 has Federation of Cancer Center (FNLCC), and substantially helped the diagnose of synovial gathered the relevant histopathologic data. Eleven sarcomas; however, it was the identification of samples of other types of soft tissue neoplasms reciprocal balanced X;18 translocations were further included in the study as the negative (p11.23;q11.2) and the resulting SS18-SSX fusion control group. These samples comprised two that revolutionized the synovial sarcoma benign (one neurofibroma, one elastofibroma) world and opened new horizons into its and nine borderline or malignant (three malignant pathogenesis and diagnosis. This rearrangement mesotheliomas, two leiomyosarcomas, two der- can be detected by fluorescent in situ hybridization matofibrosarcoma protuberans, one malignant (FISH) and reverse transcriptase polymerase chain peripheral nerve sheath tumor, and one myxoid reaction (RT-PCR). The former method is not fibrosarcoma) cases. only simpler to perform but also more practicable The study was directed in accordance with the and reproducible because the latter requires RNA declaration of Helsinki and its following revisions extraction from formalin fixed paraffin embedded and approved by the Vice-Chancellery for tissue material; this is by itself a challenge in Research Affairs of Shiraz University of Medical molecular diagnostics laboratory.1-3 Sciences (No. 8779-01-01-93). The function of the SS18-SSX fusion protein is yet to be fully elucidated; however, there are FISH procedure two common break points on Xp11 corresponding We performed FISH on tissue microarrays to SSX1 (two-thirds of cases) and SSX2 (one- containing corresponding tissues from all subjects third of cases) genes. These breakpoints join the and controls using the Zytovision dual color SS18 gene on 18q11. Interestingly, the SSX1 and break apart rearrangement probe (SS18) (Product SSX2 breakpoints and their representing fusion No. Z-2097, Zytoision company, Germany) gene have been associated with biphasic and according to the manufacturer’s instructions. monophasic synovial sarcomas, respectively. Briefly, 4-μm thick sections were Infrequently, the SSX4 or other more rare deparaffinized by placing in a 70°C oven and breakpoints, also located on Xp11, are involved.1-2 rinsing in two containers of xylene (10 min for We investigated the presence of SS18-SSX each container). Subsequent hydration was done rearrangement by FISH method in patients with in consecutive baths of 100%, 100%, 90%, and a prior diagnosis of synovial sarcoma in the 70% ethanol, each for 5 min. Pretreatment and referral centers of southern Iran. To our knowledge proteolysis were carried out by boiling the slides and based on the Medline and Scopus databases, for 15 min in prewarmed heat pretreatment this is the first report from an Iranian population. citric solution at 98°C. This was followed by the subsequent incubation of tissue sections with Materials and Methods pepsin solution for 10 min at 37°C in a humidity Subjects and design chamber. After dehydration of tissue sections by We conducted this cross-sectional study on ethanol series of 70%, 90%, and 100% degrees 23 patients diagnosed with synovial sarcoma and (1 min for each alcohol step), 10 μl SS18 probe

Middle East J Cancer 2019; 11(4): 476-482 www.SID.ir477 ArchiveAkbar Safaei of et al.SID was applied, and the slides were denatured at Chromogenic in situ hybridization (CISH) 75°C for 10 min on a hot plate. Next, the slides procedure were transferred to a humidity chamber and We performed dual-color break-apart CISH hybridized overnight at 37°C. The following day, for detecting SSX-SS18 rearrangement; however, after hydration in graded alcohol, 30 μl of it did not lead to interpretable results. DAPI/antifade-solution was administered onto the slides and incubated in the dark until Statistical analysis evaluation. Statistical analyses were performed by the Statistical Package for the Social Sciences Interpretation of FISH software version 16.0 (SPSS Inc., Chicago, IL, SS18 probe mixture contained two probes USA). Group comparisons of categorical variables hybridizing to the distal and proximal of the were analyzed using the Pearson’s chi-square breakpoint on the 18q11.2 band. These probes test. All tests were two-sided, and P values <0.05 were labeled by orange and green fluorochromes, were considered to be statistically significant. respectively. Normal cells and those tumoral cells which lacked a translocation involving the 18q11.2 Results band were expected to show two 14 (69.9%) male and nine (39.1%) female green/yellow/orange fusion signals. On the other synovial sarcoma patients participated in this hand, interphases harboring a translocation showed study. The mean age (±SD) at diagnosis was 36.4 one orange/yellow/green fusion signal, one (±15.08), ranging from 18 to 65 years. Table 1 separate green signal, and one separate orange summarizes the tumor characteristics. In addition, signal. It was expected that the distal probe hyalinization, calcification, and prominent mast hybridized to the X in rearranged cell infiltration were present in two (8.7%), eight cells, such that its corresponding orange signal (34.7%), and five (21.7%) tumors, respectively. separated from the green one which stays on the Figure 1 shows the different histological patterns 18 chromosome. of synovial sarcoma in our patients.

Immunohistochemistry Table 2 summarizes the immunohistochemical

Figure 1. The images show different histological patterns of synovial sarcoma in our patients. Biphasic (left), monophasic (middle), and poorly differentiated (right) (H & E staining, 100×).

478 Middle East J Cancer 2019;www.SID.ir 11(4): 476-482 Archive of SID SS18-SSX Rearrangement in Synovial Sarcoma

Table 1. Summary of tumor characteristics Number Frequency (%) Tumor Type Biphasic 11 47.8 Monophasic 9 39.1 Poorly-differentiated 3 13.0 Tumor Behavior Localized 10 43.5 Recurrent/Metastatic 13 56.5 Site Lower extremity 11 47.8 Internal organs 4 17.4 Head and neck 3 13.0 Upper extremity 3 13.0 Chest wall and axillae 2 8.7 Size <5 cm 8 34.8 ≥5 cm 15 65.2 Mitotic activity Score 1 (0-9 per 10 HPF) 3 13.0 Score 2 (10-19 per 10 HPF) 9 39.1 Score 3 (≥20 per 10 HPF) 11 47.8 Necrosis Score 0 (no necrosis) 11 47.8 Score 1 (<50% tumor necrosis) 12 52.2 Score 2 (≥50% tumor necrosis) 0 0.0 FNCLCC Grading II 16 69.6 III 7 30.4

characteristics of tumor cells, which were available case revealed three separate green and three in medical records for 16 patients. Epithelial separate orange signals suggestive of polyploidy; markers (cytokeratin and/or EMA) were expressed another case showed an unexpected signal pattern in about 80% of tumors. CD99, Bcl-2, and S100 consisting of an orange/yellow/green fusion signal were also reactive in 86.7%, 100%, and 37.5%, and an isolated orange one (Figure 2). respectively. All cases revealed high proliferation Among the SS18-SSX rearranged cases, 11 activity with a mean ki67 index of 35% (range, (64.7%) were males and six (35.3%) were females. 30%-80%). Moreover, one case presented focal We collected nine (52.94%) specimens from desmin staining. primary tumor sites, seven (41.18%) from the recurrence sites, and one (5.88%) from the FISH results metastatic site. 13 (76.47%) and four (23.53%) All cases had bright and interpretable signals. tumors were grade II and grade III, respectively. 17 (74%) cases revealed SS18 rearrangement, These factors, along with size, mitotic activity, but the remaining six (26%) cases showed intact and necrosis, were not statistically different from signals, thereby failing to show SS18 those of tumors without rearrangement. On the rearrangement in FISH study. All control other hand, tumor type was significantly associated specimens were also intact. Most positive with the presence of rearrangement (Chi2 test, specimens showed the expected signal pattern P=0.002) (11 biphasic and six monophasic tumors (one orange/yellow/green fusion signal, and two in rearranged group versus three monophasic and separate green and orange signals); however, one three poorly differentiated tumors in non-

Middle East J Cancer 2019; 11(4): 476-482 www.SID.ir479 ArchiveAkbar Safaei of et al.SID

Table 2. An overview of all cases with the emphasis on the immunohistochemical characteristics of tumor cells Case Type Epithelial CD99 Bcl2 CD34 Muscle S100 SS18-SSX markers markers Fusion (CK and/or EMA) (SMA/Desmin) 1 B NA NA NA NA NA NA P 2 B NA NA NA NA NA NA P 3 M + + + - - - P 4 M - + + - - - P 5 B + NA NA NA NA NA P 6 B + NA NA NA NA NA P 7 B + NA + - NA Focal P 9 M + + + - - - P 11 M NA NA NA NA NA NA P 13 M NA NA NA NA NA NA P 14 B + - - - NA NA P 15 B + + + NA NA NA P 16 M + + + - - - P 19 B + + - NA Focal Desmin NA P 21 B + + NA - - - P 22 B NA NA NA NA NA NA P 23 B + + NA NA NA NA P 8 M - + NA NA NA NA N 10 PD + - + - NA NA N 12 PD NA NA NA NA NA NA N 17 M NA NA NA NA NA NA N 18 M - + + NA - Focal N 20 PD + + + - - Focal N Total Percent 81.2% 86.7% 100% 0.0% 4.3% 37.5% B, biphasic; CK, cytokeratin; EMA, epithelial membrane antigen; M, monophasic; N, negative; NA, not available; P, positive; PD, poorly differentiated.

rearranged group). Interestingly, all tumors with Discussion prominent mast cell infiltration showed the The primary purpose of this study was to rearrangement. investigate the presence of SS18-SSX rearrangement in tumors with diagnoses synovial

Figure 2. The images show FISH results. Negative (left), positive (middle), and positive with one missed green signal (right).

480 Middle East J Cancer 2019;www.SID.ir 11(4): 476-482 Archive of SID SS18-SSX Rearrangement in Synovial Sarcoma

Table 3. Studies on molecular and conventional cytogenetic diagnosis of synovial sarcoma Country, Year N FISH CISH RT-PCR Karyotype Surace C7 Sweden and Italy, 2004 28 28/28 (100%) - 28 (100%) 27/28 (96.42%) Romeo S6 Italy, 2004 15 15/15 (100%) - 8/8 (100%) 1/1 (100%) Tvrdik D5 Czech Republic, 2005 7 - - 7/7 (100%) - Terry J11 Canada, 2005 23 22/23 (96%) Weak result - - Amary MFC10 Brazil, 2007 132 87/101 (86%) - 126/131 (96%) - Tanas MR9 USA, 2010 32 31/32 (96.87%) - - - Motoi T4 Japan, 2010 16 16/16 (100%) 16/16 (100%) 16/16 (100%) - Horna H 8 Germany, 2014 9 8/9 (89%) - 6/6 (100%) - N, Number of cases; FISH, fluorescent in situ hybridization; CISH, chromogenic in situ hybridization; RT-PCR, reverse transcriptase polymerase chain reaction.

sarcoma. This rearrangement was previously methods were selected; therefore, there was a reported in 89% to 100% of synovial sarcomas systematic error towards higher positive FISH using FISH method.4-9 However, our positive rate rates. In contrast, we performed FISH in all cases was lower. In fact, break apart FISH probe with a report of synovial sarcoma. identified 17 (73.91%) rearranged tumors, but False negative FISH results may be the remaining six (26.09%) showed intact signals. occasionally due to inherent technical difficulties. Table 3 summarizes the results of similar studies A neoplasm sometimes harbors a cryptic published on the importance of cytogenetic or molecular fusion gene, which is not detectable molecular diagnosis of synovial sarcoma. by FISH method. In such cases, a secondary Tumors that did not show SS18-SSX laboratory technique such as reverse transcriptase rearrangement were of either monophasic or polymerase chain reaction might be conducive poorly differentiated type. These types of synovial to find the rearrangement. In addition, our sarcoma can be difficult to distinguish from other understanding of the molecular alterations of tumors based solely on histomorphology and synovial sarcoma is currently incomplete, and immunohistochemistry. Four of our negative cases there still exist a large number of rare variant (cases 8, 10, 18, and 20 in table 2) had immuno- translocations linked to this entity. histochemistry information; however, only one Two cases showed unexpected FISH signal showed typical immunohistochemistry findings patterns. One revealed three separate green signals of synovial sarcoma (case 20 with positive accompanied by three separate red signals epithelial markers, CD99, Bcl6, and negative suggestive of trisomy. Since no CD34). However, not all rearranged tumors were orange/yellow/green fusion signal was detected typical in immunohistochemistry either. For in this case, we conclude that all three available instance, case 4 tumor cells did not express alleles were rearranged. The other unusual case epithelial markers, but they were rearranged for revealed one orange/yellow/green fusion signal SS18-SSX. We believe that synovial sarcoma, along with a single orange one in almost all tumor with its wide range of histological variations and cells. This pattern was also present in many other considerable immunohistochemical overlapping cases, but only in occasional scattered cells. markers is one of the most difficult sarcomas to Amary et al. reported this finding in six patients. diagnose precisely. Moreover, there is no gold They associated this result to the fact that the standard for diagnosing synovial sarcoma; SSX-SS18 transcript on was therefore, the possibility of an incorrect primary more unstable than the SS18-SSX transcript on diagnosis should always be considered. chromosome X.10 Another possible reason for the higher negative We further tried to detect SSX-SS18 FISH rate could be our method of case selection. translocation by CISH technique; however, the In some studies, only cases with a previously interpretation of CISH slides was impossible confirmed SS18-SSX fusion gene by some other mainly due to the weak signals and many lost

Middle East J Cancer 2019; 11(4): 476-482 www.SID.ir481 ArchiveAkbar Safaei of et al.SID dots in most cells. Terry et al. also had this 2015;5:7. doi: 10.1186/s13569-015-0021-3. experience with CISH and were not able to obtain 7. Surace C, Panagopoulos J, Palsson E, Rocchi M, acceptable dots. However, Motoi et al. observed Mandahl N, Mertens F. A novel FISH assay for SS18– SSX fusion type in synovial sarcoma. Lab Invest. that FISH and CISH had similar diagnostic 2004;84:1185-92. doi: 10.1038/labinvest.3700142. utilities.4, 11 8. Horna H, Allmanritter J, Doglionid C, Marxe A, Müllerb J, Gattenlöhnerf S, et al. Fluorescence in situ Conclusion analysis of soft tissue tumor associated genetic alterations in formalin-fixed paraffin-embedded tissue. We believe that FISH is a practicable method Pathol Res Pract. 2014;210:804-11. doi: for confirming the diagnosis of synovial sarcomas, 10.1016/j.prp.2014.09.009. particularly in tumors with monophasic and poorly 9. Tanas MR, Rubin BP, Tubbs RR, Billings SD, Downs- differentiated subtypes with vague immunohis- Kelly E, Goldblum JR. Utilization of fluorescence in tochemical findings. Furthermore, it is logical to situ hybridization in the diagnosis of 230 mesenchymal neoplasms. Arch Pathol Lab Med. 2010;134:1797- perform another molecular method to examine 803. doi: 10.1043/2009-0571-OAR.1. equivocal, unexpected, or discrepant FISH results. 10. Amary MFC, Berisha F, Bernardi FDC, Herbert A, James M, Reis-Filho JS, et al. Detection of SS18- Acknowledgement SSX fusion transcripts in formalin-fixed This research study was extracted from the paraffin-embedded neoplasms: analysis of conventional RT-PCR, qRT-PCR and dual color FISH as diagnostic thesis of Dr. Elham Jannesar as part of her tools for synovial sarcoma. Mod Pathol. 2007;20:482- fulfillment to obtain her speciality degree in 96. doi: 10.1038/modpathol.3800761. pathology from Shiraz University of Medical 11. Terry J, Barry TS, Horsman DE, Hsu FD, Gown AM, Sciences, Shiraz, Iran. Huntsman DG, et al. Fluorescence in situ hybridization for the detection of t(X;18)(p11.2;q11.2) in a synovial sarcoma tissue microarray using a break apart-style Conflict of Interest probe. Diagn Mol Pathol. 2005;14:77-82. doi: None declared. 10.1097/01.pas.0000155021.80213.c9.

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